THE LOCATION OF THE DONNAN FREE SPACE IN DISKS OF
BEETROOT TISSUE
By M. G.
PITMAN*
[Mamt8Cript received December 14, 1964]
Summary
This paper describes experiments which show that the cell walls of beetroot
tissue contain sufficient cation·exchange sites to account for at least 95% of the
Donnan free space (D.F.S.) as measured by Briggs, Hope, and Pitman (1958). The
contribution of the cytoplasm to the D.F.S. in their measurements was therefore
less than 5%. The exchange sites in the D.F.S. of the tissue and in the cell walls
have the same pKa of about 2·8, and are considered to be due to bound Ilronic acids.
1.
INTRODUCTION
Briggs, Hope, and Pitman (1958) measured the uptake of ions by beetroot disks
at 2°C, and showed that the free space exchange was made up of two components.
One, the water free space, was a region in which ions reached the same concentrations
as in the external solution. The other, the Donnan free space (D.F.S.), contained
fixed anions and so behaved as a cation-exchange system. The location of these phases
was not determined, though it can be shown by radioautographs of beet disks that
three-quarters of the water free space is due to cut cells at the periphery of the disk
(15% of the tissue) and the remaining quarter (5%) can be accounted for by intercellular spaces.
A cation-exchange system as part of the free space is a general property of plant
tissues, and most would agree that the exchange sites are bound uronic acids in the
cell walls. Dainty and Hope (1959) and Dainty, Hope, and Denby (1960) have shown
that this phase in Ohara australis is located in the cell walls, and that the exchange
sites are pectins or uronic acids. Crooke, De Kock, Knight, and MacDonald, in
various publications (e.g. Knight et al. 1961), have shown for many plants that
"cation-exchange capacity" can be related to uronic acid content. Moreover, simple
experiments such as the uptake of cationic dyes show that the cell wall contains a
large amount of cation absorption sites. Whether or not it behaves as a classical
Donnan phase depends on the distances between the charges in the cell wall matrix
(Dainty and Hope 1959).
It was suggested by Briggs, Hope, and Pitman (1958) that the D.F.S. was
located in the cytoplasm, which is contrary to the above observations. Even if
beet cell walls are a cation-exchange system, the method of estimation of D.F.S.
by Briggs and co-workers could have included some uptake of isotope to the cytoplasm. This paper therefore describes some measurements of ion exchange in beet
tissue and in a cell wall preparation that are designed to test the validity ofthe assump-
tion that D.F.S. is equivalent to cell wall exchange. t
* Department of Botany, University of Adelaide.
t
Reference to the present experiments was made by Dainty and Hope (1959).
Aust. J. Bioi. Sci., 1965, 18, 547-53
548
M. G. PITMAN
II.
EXPERIMENTAL AND MATERIALS
(a) Tissue Preparation
Cylinders of beetroot tissue were cut with a cork-borer 1·4 cm in diameter
and aerated in distilled water until fully turgid. They were then divided into two
portions. One set of cylinders was cut into disks 1 mm thick and aerated in distilled
water for about 3 days; the other sample was plasmolysed to separate the protoplasts
from the cell walls, and then frozen to give the tissue sufficient rigidity to be cut into
sections about 100 flo thick, which were used for the "cell wall preparation." The
average diameter of beet cells is about 100 flo, so nearly all the cells in these thin sections were cut, and lost their contents when rinsed in distilled water. Microscopic
examination showed that tissue treated this way contained very little cytoplasm,
and so reasonably can be treated as representative of the properties of cell walls.
(b) Isotopes
The isotopes used were 42K, 24Na, and 131!. 42K and 24Na were received as
irradiated K 2C0 3 or Na2C03, and were converted to the chlorides by solution in
excess hydrochloric acid. This solution was twice evaporated to dryness and the solids
redissolved in distilled water. 131J was received from Amersham as iodide in weak
thiosulphate solution (IBS 1), and was diluted with carrier iodide before use.
(c) Estimation of Exchangeable Cation or Anion in the Ti88ue
The amounts of exchangeable cation and anion in the tissue at 2°C were
estimated by the method described by Briggs, Hope, and Pitman (1958). Briefly,
uptake of isotope by disks of tissue was measured at intervals over about 4 hI' after
addition of the disks to a radioactive solution. At 2°C the fluxes of ions into the
cytoplasm were very slow (Pitman 1963) and after the rapid initial uptake, there was
little further uptake by the tissue, so that the initial uptake could be estimated by
extrapolation to t = O. The tissue was first brought to equilibrium with the nonradioactive solution by a series of exchanges which also removed exchangeable,
divalent cations.
This estimate of free space is arbitrary but the error due to other phases in the
tissue (cytoplasm) is small. Good agreement was found between this method of
determining free space exchange and a method based on elution of isotope from labelled
tissue.
(d) E8timation of Exchangeable Cation on Anion in Cell Wall Preparations
The cell wall preparation was also treated to remove exchangeable divalent
cations and equilibrate the exchange phase with the experimental solution. In a
determination, the preparation was removed from solution by filtration through
terylene toile (which is non-absorbent) and then pressed between sheets of acidwashed paper and toile to remove as much water as possible. The pellet of tissue
was then put into the experimental solution labelled with 131J (as an anion) and the
sections resuspended. After 1 hI' the sample was again separated from the solution,
pressed, and weighed.
549
DONNAN FREE SPACE IN BEETROOT TISSUE
The 13 11 was extracted from the sample by a series of exchanges with unlabelled
salt solution, and used to estimate the "iodide free space" (I.F.S.). The I.F.S. was
due mainly to solution unavoidably included in the sample with the cell walls. Assuming the weight and volume of the I.F.S. were equal, the difference between weight
of sample and I.F.S. then gave an estimate of the weight of this particular sample
that was not accessible to iodide.
After the 131 1 determination the sample was resuspended in solution and the
uptake and elution process repeated using potassium or sodium isotopes. The amount
of potassium or sodium in the sample was mainly due to the cell wall exchange, but
included some in solution taken up with the sample. This solution was estimated as
the difference between sample weight and the previously determined weight
inaccessible to iodide. Hence the exchangeable potassium or sodium due to nonexchangeable anions could be determined (extra-exchangeable potassium or sodium).
TABLE
1
COy[PARISON OF EXCHAXGEABLE POTASSIUM IN DISKS OF BEETROOT TISSUE A='iD IN CELL WALL
PREPARATIONS FROM THE SAME TISSUE
Disks of Tissue
Cell Wall Preparation
--------
Heplicate
No.
Sample
Weight
(g)
Extraexchangeable
Potassium
(m-equivjkg)
"
I Equivalent
I
Fresh
Weight
(g)
Weight
Inaccessible
to Iodide
(g)
i
3·30
0·37
-[
0·37
I
10·4
0·33
I
10·4
Replicate I
No.
I
-1----·I
1·92
9·8
2
1·84
10·1
2
3·15
3
1·91
10·0
3
3·05
Mean
I
I --_.__._-
9·9±0·1
_.--------
III.
.._ - - -
[
Extraexchangeable
Potassium
(m-equivjkg)
10·0
I
[~3±0'1-
------
EXPERIMENTAL RESULTS
(a) Comparison of Extra-exchangeable Potassium and Sodium
The results of a comparison of exchangeable potassium in tissue and in a cell
wall preparation are given in Table 1. Three replicates of each were used and there
is satisfactory agreement both between replicates, and between the means of each
series, i.e. the D.F.S. of the disks appears to be completely equivalent to cell wall
exchange.
The results of Table 1 were obtained with a potassium chloride solution of
concentration 5 m-equiv 11. In Table 2 are similar estimates, but over a wider range of
concentration for both potassium and sodium. In both tissue and cell wall preparation there is good agreement between potassium and sodium concentration and no
sign of specific uptake. The difference between the tissue and cell wall exchange was
greater, amounting to 0,5-1·0 m-equiv/kg at the higher concentrations. This
difference could be due to uptake of isotope to the cytoplasmic phase which would
550
M. G. PITMAN
appear in the tissue D.F.S. estimate, but not in the cell wall estimate. Unfortunately
there is an uncertainty in comparing the two results as it is difficult to estimate the
fresh weight of tissue equivalent to a particular cell wall sample. This was done by
measuring the length of beet cylinders before and after freezing, and then using all
the sections cut from a known length of the frozen tissue (about 1 cm). It is considered
that the error arising in this way could be about 5% which is larger than the experimental error and standard error of the mean of Table 1. In spite of the good agreement between the sets of data given in Table 1 it can be concluded that the D.F.S.
and cell wall exchange agree only to within 5%.
TABLE
2
EXTRA-EXCHANGEABLE SODIUM OR POTASSIUM IN DISKS OF BEETROOT TISSUE AND IN CELL WALT.
SAMPLES IN EQUILIBRIUM WITH SOLUTIONS OF VARIOUS CONCENTRATIONS
Disks of Tissue*
Solution
Concentration
(m-equiv/l)
Extraexchangeable
Potassium
(m-equiv/kg)
Extraexchangeable
Sodium
(m-equiv/kg)
5
11·0
11.·2
10
12·3
12·4
15
13· L
20
12·!J
!
Cell Wall Samplest
Extraexchangeable
Potassium
(m-equiv/kg)
Extraexchangeable
Sodium
(m-equiv/kg)
12·0
12·0
10·5
11·5
13·4
12·0
ll·O
13·5
12·5
11·5
I
I
* Estimated experimental error ±O· 5 m-equiv/kg tissue.
t Estimated experimental error within the same sample = 0·5 m-equiv/kg tissue.
(b) Effect of pH on Extra-exchangeable Potassium
From the change in extra-exchangeable potassium with concentration, Briggs,
Hope, and Pitman (1958) suggested that the pKa of the anions in the D.F.S. was
about 2·8. This suggestion has been supported by the measurement of the extraexchangeable cation in beet disks as a function of pH when the concentration of the
potassium in solution was 10 m-equiv(l. The results are shown in Figure 1 as points,
the line representing the expected values if pKa = 2 ·8, [KoJ = 10 m-equiv(l, and
non-exchangeable anion concentration = 500 m-equiv(l.*
* The calculation of the relationship between Donnan exchange, salt concentration, and
external pH is given by Dainty, Hope, and Denby (1960). Briefly,
Na.
[
.]
=
!{[Ka 2[Na oJ2
-
[HoP
4K ao<[Na o]2]l _ Ka[Na o]}
[Ho] ,
+ [Ho]([Ho]+[NaoJ)
where [Nat], [Na o], [Hi]' and [Ho] are concentrations of ions inside and outside the Donnan phase
respectively, Ka is the dissociation constant, and 0< is the total amount of non-exchangeable anion,
dissociated and undissociated.
551
DONNAN FREE SPACE IN BEETROOT TISSUE
The degree of dissociation of the Donnan anions, and hence the amount of
extra-exchangeable cation, is determined by the [H +] in the phase. This in turn is
dependent on external [H +] and also on total cation content of the solution, for
hydrogen ions are distributed between solution and Donnan phase like any other
cation. As a result the pH of the solution producing 50% dissociation will not usually
be equal to the pK a. In the example given above, 50% dissociation occurs at pH
4 ·19 in a KGI solution of concentration 10 m-equivjl, but at lower concentrations
the apparent pKa shifts to higher values, e.g. in a 1 m-equivjl solution it would occur
at a pH of 5·19.
10
~
:<
?'
:>
aOJ
B
I
,3
:>'
:>
iii
III
6
«
fo
"-
OJ
..I
OJ
«
4
OJ
"«z
:r
u
x
OJ
~
2
II:
fX
OJ
O~\
______ ______ ______ ______ ______ ______
~
~
~
~
~
~
pH
Fig. I.-Effect of pH on extra-exchangeable potassium in beetroot
disks. Concentration of potassium in solution was 10 m-equivJI. The
line gives values calculated for pKa of 2·8.
The effect of pH on exchangeable potassium or sodium also has been investigated
for the cell wall preparation, but in this case it was possible to estimate the amount
of exchangeable cation from titration with hydrochloric acid. This titration was
carried out with solutions that were all 10 m-equivjl with respect to sodium, to
prevent drift in the apparent pKa during titration. (Thus the acid solution was
5 m-equiv/l HGI + 10 m-equivjl NaGl.) A sample of cell wall preparation was first
equilibrated (as described above) with a solution of sodium chloride of concentration
10 m-equivjl, pressed, and the pellet suspended in 50 ml sodium chloride solution
of concentration 10 m-equivjl. Sodium hydroxide (10 m-equivjl, 5 ml) was added to
turn all the exchange sites to the ionized form and then the system titrated.
In Figure 2, A, is plotted the difference in titre over a range of pH values
between this system and 5 ml of sodium hydroxide alone. Points below the zero line
are exchange sites which were un-ionized under the conditions of preparation. In
sodium chloride solution of concentration 10 m-equivjl, the apparent pKa of the
weak acid in the cell walls responsible for most of the cation exchange was about
552
M. G. PITMAN
4·0, and is in good agreement with the value found for the tissue exchange sites
(4·2, Fig. 1).
A similar titration is shown in Figure 2, B, but in this case the cell wall preparation was first treated with dilute acid at lOoDe for 15 hr. Other less extreme treatments (e.g. boiling water) had little or no effect on the cation-exchange system .
10
•
•A
•
•
:;
6
0
u
<:
u
"'0
...J
I
U
•
',."
0
I
IL
0
w
::i
OJ
...J
0
•
•
•
5
0
>
•
•
•
0
•
•
•••••••
• ••••
•
•
•
•
ooB
••••
0°
.00000000000000000000000000
·0
-51
11
10
I
I
I
9
8
7
pH
I
I
I
6
5
4
I
3
2
Fig. 2.-Tit.ration of cell wall preparation; titre of hydrochloric
acid (conen. 5 m-equivjl) t·. pH for cell wall preparation (A) and
for the same preparation after hydrolysis with dilute acid at 100°0
for 15 hr (B).
IV.
DISOUSSION
It is considered that the results given in this paper show that the cell wall is a
cation-exchange system and responsible for the D.F.S. Firstly, there is good agreement between estimates of non-exchangeable anion in both tissue and cell wall
preparation. Secondly, the pKa of the weak acid responsible for the cation exchange
system is the same in cell walls and in the tissue D.F.S. Any other contribution to
the D.F.S. is small: at lower concentrations (5 m-equiv/l) the difference between
cell walls and tissue is negligible, but at higher concentrations it may be as large as
5-10% of the D.F.S. This difference could be due to the cytoplasmic phase in the
tissue, as the flux into this phase (which determines the isotope uptake) is more or
less proportional to solution concentration (Pitman 1963).
The exchange sites in the cell walls are only removed by extreme treatments.
The amount of exchange in the walls was not affected by boiling water, and hydrolysis
at lOoDe for 15 hI' in dilute sulphuric acid was necessary to remove a substantial
DONNAN FREE SPACE IN BEETROOT TISSUE
553
proportion of the exchange sites. Knight et al. (1961), investigating polygalacturonic
acids in storage tissues, also found that most of the cell wall content was not extracted
by water, and estimated that about 10% of the dry weight of the walls was uronic
acid. In the present experiments, 1 g tissue gave 18 mg (dry wt.) of cell wall containing 10 fL-equiv. of exchange sites, which, if taken to be uronic acid, is 1·8 mg
dry weight, i.e. these exchange sites were also about 10% of the wall dry weight.
It seems reasonable to assume that the exchange sites in beet cell walls are due to
relatively tightly bound uronic acids.
The concentration of non-exchangeable anions in the D.F.S. was estimated by
Briggs, Hope, and Pitman (1958) to be about 500m-equiv/1 (derived from lOm-equiv/
kg in about 20 ml per 1 kg tissue). The volume of the cell wall preparation not
accessible to iodide was about 110 ml/kg (Table 1). As the dry weight of the walls
was 20 g/kg, the volume of the D.F.S. would have been about 90 (i.e. 110-20)
ml/kg,* if cell wall not free to iodide were D.F.S. The cell wall components thus
appear to be binding about three times their weight of water in a region not
accessible to ions.
V. ACKNOWLEDGMENTS
This work was done at the Botany School, University of Cambridge.
also grateful to Dr. A. B. Hope for his helpful comments.
VI.
I am
REFERENCES
BRIGGS, G. E., HOPE, A. B., and PITMAN, M. G. (1958).-Exchangeable ions in beet discs at low
temperature. J. Exp. Bot. 9: 128-41.
DAINTY, J., and HOPE, A. B. (1959).-Ionic relations of cells of Ohara australis. 1. Ion exchange
in the cell wall. Aust. J. Biol. Sci. 12: 395-411.
DAINTY, J., and HOPE, A. B. (196l).-The electric double layer and the Donnan Equilibrium in
relation to plant cell walls. Aust. J. Biol. Sci. 14: 541-51.
DAINTY, J., HOPE, A. B., and DENBY, C. (1960).-Ionic relations of cells of Ohara australis.
II. The indiffusible anions in the cell wall. Aust. J. Biol. Sci. 13: 267-76.
KNIGHT, A. H., CROOKE, W. M., MACDoNALD, 1. R., and SHEPHERD, H. (1961).-Changes in the
pectin (uronic acid) content of storage tissue discs. J. Exp. Bot. 12: 13-26.
PITMAN, M. G. (1963).-The determination of the salt relations of the cytoplasmic phase in cells
of beetroot tissue. Aust. J. Biol. Sci. 16: 647-68.
* Assuming 20 g/kg of cell wall =
20 mljkg.